The influenza virus is a membrane-enclosed RNA virus, whose genome consists of discretized segments of negative-sense RNA, and is primarily an infectious agent of avians and humans (Lamb et al., “The Gene Structure and Replication of Influenza Virus,” Annu Rev Biochem 52:467-506 (1983)). The influenza proteins responsible for docking, hemagglutinin (HA), and budding, neuraminidase (NA), are anchored in the viral lipid membrane and are the major antigenic focus of the prevention and treatment of infection. There are three distinct genera of the influenza virus—denoted A, B, and C, and typified by differences in structural proteins—that cause immunological effects of variable severity (Cox et al., “The Molecular Epidemiology of Influenza Viruses,” Semin Virol 6:359-370 (1995)), such as the typical symptoms associated with the flu (Monto et al., “Clinical Signs and Symptoms Predicting Influenza Infection,” Arch Intern Med 160:3243-3247 (2000)). Influenzas A and B are the most immunologically relevant due to their co-circulating seasonal infectivity in human populations (Lin et al., “Recent Changes Among Human Influenza Viruses,” Virus Res 103:47-52 (2004)), and influenza A has been responsible for all recorded pandemics. Influenza A infects many different animal species besides humans, including ducks, chickens, pigs, whales, horses, and seals. Influenza B viruses generally only infect humans.
All three types of influenza virus have genomes composed of eight different RNA helices, which encodes a single gene and are bound by a nucleoprotein that determines the viral type: A, B, or C. In effect, the influenza genome is made up of eight separate pieces of nucleic acid that can come together to form viruses with new combinations of viral genes when cells become co-infected by more than one viral type. Two of these RNA helices encode the important viral surface proteins hemagglutinin and neuraminidase, which are embedded in the lipid bilayer of a mature virus particle.
Variations in the viral hemagglutinin and neuraminidase determine the viral subtype. Hemagglutinin is responsible for entry of the virus into the host cell, while neuraminidase is important in the release of newly formed viruses from the infected cells. Antibodies to hemagglutinin can neutralize the virus and are the major determinant for immunity. Antibodies to neuraminidase do not neutralize the virus but may limit viral replication and the course of infection. Host antibodies to specific types of hemagglutinin and neuraminidase prevent and generally ameliorate future infection by the same viral strain. However, since the genetic makeup of viral strains is dynamic and ever-changing, immunity gained through successful resistance to one strain gained during an infection one year may be useless in combating a new, recombined, variant strain the next year.
Epidemics of influenza are thought to result when viral strains change over time by the process of antigenic drift. Antigenic drift (caused by mutations in the principal viral antigen genes, especially in the hemagglutinin or neuraminidase genes) results in small changes in surface antigens, and occurs essentially continuously over time. When these changes occur in the right places in the genes, they render the new antigens unrecognizable by the antibodies raised against other influenza virus strains during previous infections.
Influenza pandemics (or worldwide epidemics) occur as a result of “antigenic shift.” Antigenic shift is an abrupt, major change in an influenza A virus that results from a new hemagglutinin and/or new hemagglutinin and neuraminidase protein appearing in an influenza A strain. Such shifts are generally thought to occur when a new combination of viral genomic RNAs is created, possibly in a non-human species, and that new combination is passed to humans. When such an antigenic shift occurs, most humans have little or no protection against the virus, and an infection can prove lethal.
Periodically, unique strains of the influenza virus may emerge, and the antigenic novelty of the pathogen results in enhanced rates of infectivity, transmission, and morbidity. History has shown that these influenza pandemics may be extremely deadly. For example, the “Spanish Flu” (Johnson et al., “Updating the Accounts: Global mortality of the 1918-1920 “Spanish” Influenza Pandemic,” Bull Hist Med 76:105-115 (2002)) (H1N1; 1918-1920 upwards of 50 million deaths), the “Asian Flu” (Rajagopal et al., “Pandemic (avian) Influenza,” Semin Respir Crit. Care 28:159-170 (2007)) (H2N2; 1957-1958, 1 million deaths), and the “Hong Kong Flu” (Shalala et al., “Collaboration in the Fight Against Infectious Diseases,” Emerg Infect Dis 4:354-357 (1998)) (H3N2; 1968-1969, 700,000 deaths) demonstrated the efficiency of the virus and its ability to quickly spread worldwide; additionally, the rapidity in which infectious strains may emerge, and our innate resistance to them, was clearly highlighted. To combat these pandemics, the first mono- and bivalent influenza vaccines were developed in the mid-1940s, which consisted of deactivated influenza viruses (Henle et al., “Demonstration of the Efficacy of Vaccination Against Influenza Type A by Experimental Infection of Human Beings,” J Immunol 46:163-175 (1943), Francis et al., “Protective Effect of Vaccination Against Induced Influenza A,” J Clin Invest 24:536-546 (1945), Salk et al., “Protective Effect of Vaccination Against Induced Influenza B,” J Clin Invest 24:547-553 (1945)). Trivalent influenza vaccines have since been become standard and consist of three deactivated strains (Halperin et al., “Safety and Immunogenicity of a Trivalent, Inactivated, Mammalian Cell Culture-derived Influenza Vaccine in Healthy Adults, Seniors, and Children,” Vaccine 20:1240-1247 (2002)), or three live, attenuated strains (Belshe et al., “The Efficacy of Live Attenuated, Cold-adapted, Trivalent, Intranasal Influenzavirus Vaccine in Children,” New Engl J Med 338:1405-1412 (1998))—two of influenza A and one of influenza B—in order to provide broader preventative measures against co-circulating seasonal strains. Since these initial efforts, much research has been performed towards developing improved, more enveloping vaccines based on recombinant technology to not only immunize against current viral strains (Kilbourne et al., “Future Influenza Vaccines and the Use of Genetic Recombinants,” Bull World Health Org 41:643-645 (1969), Webby et al., “Are We Ready for Pandemic Influenza?,” Science 302:1519-1522 (2003), Treanor et al., “Safety and Immunogenicity of a Baculovirus-Expressed Hemagglutinin Influenza Vaccine: A Randomized Controlled Trial,” J Am Med Assoc 297:1577-1582 (2007)), but also provide protection against past epidemic strains as well.
Avian influenza, of the H5N1 designation, is currently the subject of major international research efforts. Past influenza pandemics have proven that in the absence of proper safeguards, new and highly pathogenic strains of influenza can be extremely deadly. With the rise in the global population and the advent of international travel and commerce, the repercussions of a pandemic would be devastating. Since it was initially isolated in 1997 (de Jong et al., “A Pandemic Warning?” Nature 389:554 (1997)), there have been a reported 380 cases of H5N1 that have resulted in 240 deaths (World Health Organization, “Epidemic and Pandemic Alert and Response: Avian Influenza,” accessed online from the WHO on Apr. 16, 2008). The majority of these reported cases are transmitted from avians to humans, but isolated cases of human-to-human transmission have been reported (Ungchusak et al, “Probable Person-to-Person Transmission of Avian Influenza A (H5N1),” N Engl J Med 352:333-340 (2005)).
Very recently, there have been reports of an H1N1, type A, strain of swine influenza that has unique genetic properties and is capable of human-to-human transmission. The initial outbreak appeared in Mexico, but cases have now been reported in a number of urban centers across the United States and elsewhere in the world. As of Apr. 30, 2009, the World Health Organization has raised the Alert Level to Phase 5.
Vaccines are essential as preventative measures against disease, but traditional drug-based therapeutics are also required in the event that the vaccine supply is limited or not yet available, scenarios that are especially worrisome in highly virulent pandemic viral strains such as with avian influenza H5N1 (Kilpatrick et al., “Predicting the Global Spread of H5N1 Avian Influenza,” Proc Natl Acad Sci USA 103:19368-19373 (2006)). For example, neuraminidase, the influenza enzyme that controls the release of the newly packaged virus from the host cell membrane (Wagner et al., “Interdependence of Hemagglutinin Glycosylation Neuraminidase as Regulators of Influenza Virus Growth: A Study by Reverse Genetics,” J Virol 74:6316-6323 (2000)), is an attractive drug target in the influenza lifecycle. Oseltamivir (TAMIFLU™) (Kim et al., “Influenza Neuraminidase Inhibitors Possessing a Novel Hydrophobic Interaction in the Enzyme Active Site Design, Synthesis, and Structural Analysis of Carboxylic Acid Sialic Acid Analogues with Potent Anti-Influenza Activity,” J Am Chem Soc 119:681-690 (1997)) is an orally active antiviral that acts as a transition state mimic of the active site of neuraminidase. It is currently suggested that world centers begin stockpiling supplies of TAMIFLU™ (and other antivirals, such as Zanamivir (Itzstein et al., “Rational Design of Potent Sialidase-Based Inhibitors of Influenza Virus Replication,” Nature 363:418-423 (1993)) in the event of a sudden pandemic (Moscona et al., “Neuraminidase Inhibitors for Influenza,” New Engl J Med 353:1363-1373 (2005)). While antivirals are capable therapies, preventative rather than reactive measures will ultimately ensure long-term success against deadly influenza virus pandemics since drug-resistant forms of influenza are readily appearing (de Jong et al., “Oseltamivir Resistance During Treatment of Influenza A (H5N1) Infection,” New Engl J Med 353:2667-2672 (2005)).
An ancillary development stemming from researchers' ability to produce and amplify recombinant proteins, and the genes from which they are encoded, is the high-throughput microarray. While initial applications of high-throughput screening focused on genomic arrays (Schena et al., “Quantitative Monitoring of Gene Expression Patterns With a Complementary DNA Microarray,” Science 270:467-470 (1995), Lipshutz et al., “High density Synthetic Oligonucleotide Arrays,” Nat Genet. 21:20-24 (1999)), the protein microarray has found a variety of significant uses as well. For example, proteome profiling via protein microarrays has unveiled a myriad of novel interactions (MacBeath et al., “Printing Proteins as Microarrays for High-Throughput Function Determination,” Science 289:1760-1763 (2000), Michaud et al., “Analyzing Antibody Specificity With Whole Proteome Microarrays,” Nat Biotech 21:1509-1512 (2003), Chan et al., “Protein Microarrays for Multiplexed Analysis of Signal Transduction Pathways,” Nat Med 10:1390-1396 (2004)). Protein microarrays have been used to discover antigenic proteins and monitor human immunological responses to them (Davies et al., “Profiling the Humoral Immune Response to Infection by Using Proteome Microarrays: High-Throughput Vaccine and Diagnostic Antigen Discovery,” Proc Natl Acad Sci USA 102:547-552 (2005), Li et al., “Protein Microarray for Profiling Antibody Responses to Yersinia pestis Live Vaccine,” Infect Immun 73:3734-3739 (2005), Qiu et al., “Antibody Responses to Individual Proteins of SARS Coronavirus and Their Neutralization Activities,” Microbes Infect 7:882-889 (2005)). This tactic has not been used previously for immobilization of multiple isoforms of the influenza antigen hemagglutinin. Moreover, in each of these reports, detection was achieved using labeled reagents.
It would be desirable to provide an array of immobilized antigen isoforms that can be used to screen for antibodies against infectious agents and vaccines involving multiple similar specificities, e.g., distinguishing between different strains of an infectious agent such as influenza based on the immune response generated by these infectious agents, or vaccines against them, using unlabeled reagents. In view of the possibility of influenza pandemic, it would also be desirable to develop a system capable of screening putative vaccine therapies for efficacy and/or cross-protection against various strains of influenza. Furthermore, a system able to rapidly screen for the presence of avian influenza or other strains in wildlife and livestock would be of considerable utility in monitoring the status and spread of the disease.
The present invention is directed to overcoming these and other deficiencies in the art.